Competitive isotopic and non-isotopic immunoassays or binding assays with solid phase or second antibody separations for measuring analytes in biological fluids have been described in the literature for over two decades. Numerous United States and foreign patents have been issued dealing with one or more aspects of these basic techniques.
Competitive enzyme immunoassays for various analytes were disclosed in U.S. Pat. Nos. 3,654,090 to Schurrs et al and 3,850,752 to Schurrs et al.
In enzyme immunoassays the enzyme label is prepared by one of several methods in which the analyte is covalently attached to the enzyme and the free unreacted analyte is separated from the enzyme labeled analyte either by dialysis and/or chromatography.
In such methods the free unconjugated enzyme is, in most cases, not separated from the conjugated enzyme for two practical reasons:
1. It requires tedious affinity purification and if accomplished produces a very unstable conjugated enzyme-analyte label since the latter constitutes a very low ratio of the unconjugated enzyme. PA1 2. The analyte specific binder bound enzyme, and not the free unbound enzyme, is the fraction that is quantitated after exhaustive washing of the free unbound enzyme. PA1 a column-type assembly defining a fluid pathway having an open end adapted to receive a sample of biological fluid to be analyzed, said fluid pathway being bridged by a first solid phase support, and an effluent discharge point on the side of said support opposite said open end, PA1 a sleeve-type container having an open end and a closed end, said assembly being received in said open end of said sleeve-type container, PA1 a specific binder (Ab) covalently immobilized on said solid phase support to which an analyte label (Ag.sub.1 *) is prereacted to saturate almost all binding sites on said binder to form a first solid phase specific binder-analyte label complex, -Ab.apprxeq.Ag.sub.1 *, said solid phase complex when contacted with a biological fluid sample containing a specific analyte (Ag.sup.1), being adapted to have displaced therefrom labeled analyte (Ag.sub.1 *) in an amount directly proportional to the concentration of Ag.sup.1, PA1 a second solid support, spaced apart from first solid phase support, housed at the closed end of said sleeve-type container and in proximity to said effluent discharge point, said second solid support, when contacted by the displaced labeled analyte (Ag.sub.1 *) from the effluent discharge point said first solid phase complex, being adapted to produce a visible color on said second solid support either directly or after the addition to said second solid support a substance capable of reacting with the analyte label to produce a visible color.
It follows, therefore, that in the aforementioned type assays the zero dose concentration has the highest signal since there is no analyte to compete with the labeled analyte for binding sites on the analyte's specific binder.
In competitive enzyme immunoassays the absence of a given analyte in a sample produces the highest color while the presence of a given analyte in a sample will produce progressively less color as compared to the zero dose depending on the concentration of said analyte in a given sample. For qualitative, "on site" type assays whereby a "yes" "no" answer is needed for the detection of a given analyte in biological fluids, the type of competitive enzyme immunoassays described in U.S. Pat. Nos. 3,654,090 and 3,850,752 are unsuitable for obvious reasons: (1) the decrease in color from a reference zero dose is difficult to detect by the naked eye, and (2) washing is required to separate the free unbound enzyme from the bound enzyme.
It is the object of the present invention to reverse such a trend since it is more logical to observe the presence of color in samples containing a given analyte while negative samples, samples devoid of a given analyte, produce no color.
U.S. Pat. No. 3,817,837 to Rubinstein et al and U.S. Pat. No. 3,852,157 to Rubinstein et al and other follow-up patents disclose homogenous type enzyme amplification immunoassay for haptens where the separation of antibody bound enzyme from free unbound enzyme is not required. In said enzyme amplification assay system the antibody-hapten enzyme labeled complex inhibits the enzyme from reacting with its substrate since the active site on the enzyme molecule is sterically hindered by the antibody to the hapten. By contacting the antibody's hapten-enzyme complex and the enzyme complex and the enzyme substrate with a sample containing a given hapten to the antibody it competes with the hapten-enzyme label for antibody sites thus allowing the enzyme to react with its specific substrate. This technique is limited to few enzymes specifically glucose-6-dehydrogenase (U.S. Pat. No. 3,875,011) malate dehydrogenase (U.S. Pat. No. 4,191,613) and U.S. Pat. Nos. 4,203,802 and 4,067,774.
U.S. Pat. No. 4,434,236 to Freytag discloses a method for the rapid determination of analytes in biological specimens by using an analyte-analogue immobilized on a solid phase wherein a displaceable labeled antibody to the analyte is found. In this disclosed method the antibody has a greater affinity for the analyte than for the analyte-analogue. The presence of an analyte in a sample specific for said antibody will easily displace the labeled antibody. Consequently, the amount of displaced labeled antibody is related to the amount of analyte present in the sample.
Although this method of U.S. Pat. No. 4,434,236 is an improvement over the previously cited patents it relies on two important factors; namely, (i) the use of an analyte-analogue that has a lower affinity to the analyte's labeled antibody, and (ii) the immobilization of analyte-analogues, especially small compounds (such as haptens), on a solid support is not easily achieved and requires specific functional groups on the analyte-analogue in order to affect immobilization. Furthermore, since the affinity of the analyte-analogue to the analyte's antibody is purposely low, the changes of labeled antibody leaking off the solid phase is quite probable.
U.S. Pat. No. 4,446,232 to Liotta, is similar in context to that disclosed in U.S. Pat. No. 4,434,236, wherein a given antigen is impregnated in a given matrix in the first zone of the disclosed device. In said matrix containing a given antigen, an enzyme-linked antibody is reacted to said antigen. In the presence of antigen in a biological specimen the antibody is displaced into a second zone which contains materials capable of reacting with the enzyme linked antibodies to produce a color. Determination of antibodies in biological fluids is also disclosed by Liotta wherein the antibody is impregnated in first zone and reacted with enzyme-linked antigen. Although this approach is a simple modification of Freytag's approach, it suffers several technical drawbacks; (1) the impregnation of antigens or antibodies in the first zone of Liotta's device will be prone to antigen or antibody "leakage" in the absence of patient antigens or antibodies, (ii) the affinity of enzyme-linked antibody or antigen is not defined. The competition between sample antigen and impregnated antigen in the first zone to the enzyme-linked antibody is by no means instantaneous because of steric hindrance and the ability of the sample antigen, like hCG in Example 1 of said patent, to dislodge the enzyme-linked antibody from the impregnated antigen is highly improbable because of steric hindrance and equilibrium considerations.
It is well established in the art that large molecules (greater than 20 kilo daltons), require longer incubations with their specific binders to reach equilibrium. When the specific antibody (molecular mass 150 kilo daltons) is linked to an enzyme (molecular mass greater than 50 kilo daltons), i.e., the effective molecular mass of the enzyme-linked antibody is approximately 200 kilo daltons, and said enzyme-linked or antigen is not defined. The competition between sample antigen and impregnated antigen in the first zone to the enzyme-linked antibody is by no means instantaneous because of steric hindrance and the ability of the sample antigen, like hCG in Example 1 of said patent, to dislodge the enzyme-linked antibody from the impregnated antigen is highly improbable because of steric hindrance and equilibrium considerations.
It is well established in the art that large molecules (greater than 20 kilo daltons), require longer incubations with their specific binders to reach equilibrium. When the specific antibody (molecular mass 150 kilo daltons) is linked to an enzyme (molecular mass greater than 50 kilo daltons), i.e., the effective molecular mass of the enzyme-linked antibody is approximately 200 kilo daltons, and said enzyme-linked antibody is prereacted with an antigen (now molecular mass is approximately 200 kD) the patient antigen (20 kD) will require time to compete with the pre-reacted antigen bound to the enzyme-linked antibody and displace it. This is quite obvious from the following equilibria. ##STR1## where k.sub.1 =k.sub.2 ; k.sub.3 =k.sub.4 and k.sub.1 &gt;&gt;k.sub.3 because of steric hindrance and displacement of Ag.sub.1 from E-Ab Ag.sub.1 by Ag.sup.1 is not easily achieved as disclosed in U.S. Pat. No. 4,446,232. Furthermore, k.sub.3 &gt;K.sub.4 only if the concentration [Ag.sup.1 ]&gt;&gt;[Ag.sub.1 ] that displacement will occur. This means that the amount of Ag.sup.1 measured will only be at high concentrations; therefore, low sensitivity assay.
This fact is exemplified in European Patent Application No. 0 279 097 in which Fuerstenberg shows that Liotta's disclosure in U.S. Pat. No. 4,446,232 is quite insensitive as shown in Example 1 of EPA 0 279 097 for theophylline were the reflectance difference between theophylline levels of 10.4 ug/ml (therapeutic threshold) and 19.4 ug/ml (toxic threshold) is 0.55-0.43 or 0.12 units. Similarly, in Example 2 of EPA 0 279 097,200 mIU hCG was required to produce a change in color and displace the enzyme-linked hCG antibody. By all analytical standards the Liotta disclosure and the examples cited in EPA 0 279 097 using Liotta's method show that said method as disclosed in U.S. Pat. No. 4,446,232 is not sensitive enough to be a reliable analytical tool.
Other United States and foreign patent specifications and applications dealing with elements for the determination of biological fluids are cited here for completion.
U.S. Pat. Nos. 4,144,306; 4,366,241; 4,740,468; 4,774,192; 4,632,901; 4,774,174; 4,769,333; 4,769,216; 3,811,840 and 4,042,335.
European Patent Specification Nos. 0 042 755; 0 070 300 and European Patent Applications 0 281 201; 0 284 232 and International Applications WO84/029193 and WO88/06723